VP22融合型显性负性突变体抑制乙肝病毒复制
作者：易军，宫卫东，王岭，凌瑞，陈江浩，王辉，王廷
【关键词】 肝炎病毒
Inhibition of hepatitis B virus replication by dominant negative mutant of VP22 fusion protein
【Abstract】 AIM: To investigate the inhibitory effect on hepatitis B virus (HBV) replication with dominant negative (DN) mutant of VP22 fusion protein. METHODS: Fulllength or fractions of VP22 were fused to C terminal of HBV core protein (HBc), and cloned into pcDNA3.1(-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the concentration of HBV surface antigen (HBsAg) in the supernatant of cell culture. And MTT assay was performed to detect the cytotoxicity of transgene expression to the host cells. RESULTS: DN mutant of VP22 and its fractions could be expressed in HepG2.2.15 cells, and had no toxic effect on the host cells. The DN mutant could inhibit HBV replication, and the mutant with protein transduction ability had a stronger inhibition than that without. The DN mutant of fulllength VP22 had the strongest inhibitory effect, reducing the HBsAg concentration by 81.94%. CONCLUSION: DN mutant of VP22 fusion protein can enhance the inhibition of HBV replication.
【Keywords】hepatitis B virus; dominant negative mutant; VP22
【摘要】 目的： 了解VP22融合型显性负性(DN)突变体对抑制乙肝病毒(HBV)复制的作用. 方法： 将VP22全长及其不同区段融合于HBV核心蛋白(HBc)的C端，克隆入pcDNA3.1(-)构成DN突变体真核表达质粒. 转染HepG2.2.15细胞后，免疫荧光鉴定DN突变体在细胞内的表达，并以培养上清HBV表面抗原(HBsAg)的浓度为指标，观察DN突变体对HBV病毒复制的抑制效应. 同时以MTT比色法观察转基因的表达对宿主细胞生长状态的影响. 结果： VP22及其不同区段构建的DN突变体可在HepG2.2.15细胞中进行表达，且对宿主细胞无毒性. VP22融合型DN突变体可有效地抑制HBV的复制，具有蛋白转导特性的DN突变体比无转导特性的DN突变体具有更强的抗病毒能力. 其中VP22全长构成的DN突变体具有最强的抑制效应，可使上清HBsAg浓度下降81.94%. 结论： VP22融合型DN突变体可以增强DN突变体对HBV复制的抑制.
【关键词】 肝炎病毒，乙型；显性负性突变体；VP22
0引言
全球目前有3.5亿慢性乙型肝炎病毒（hepatitis virus B, HBV）感染者，每年约有100万人死于HBV感染的'相关疾病，占疾病死因的第9位［1］. 目前，国际上尚无有效治疗慢性乙肝的手段. 基因治疗技术的不断发展为抗HBV治疗提供了新的思路和途径. 其中显性负性(dominant negative，DN)突变体的胞内表达是HBV基因治疗的重要策略之一［2-6］. Elliott等［7］发现，单纯疱疹病毒1(herpes simplex virus type 1, HSV1)的结构蛋白VP22(及其融合蛋白)可从培养基进入细胞内，并可不经过细胞细胞接触依赖性在同型或异型细胞之间转移. 我们将HBV核心蛋白(HBV core protein, HBc)与VP22进行融合构成DN突变体，以期利用VP22的蛋白转导特性进一步提高DN突变体的抗病毒效应.
1材料和方法
1.1材料质粒pVP22/mycHis2和pcDNA3.1(-)为Invitrogen公司产品；EBOHBV克隆有1.3倍HBV全长基因组为本室保存. 限制性内切酶、连接酶购自TaKaRa Biotech Co.Ltd；鼠抗HBc抗体和鼠抗cmyc抗体为SantaCruz公司产品，FITC标记的羊抗鼠IgG为博士德生物工程有限公司产品；DMEM培养基、进